Summary: The purpose of this project is to identify and characterize virulence determinants in Corynebacterium diphtheriae in order to understand how this important pathogen causes disease. Expression of diphtheria toxin, the primary virulence determinant of C. diphtheriae, is regulated by iron, and it is likely that additional virulence factors are coordinately regulated with that of the toxin. The ability to acquire iron during an infection is essential for many bacterial pathogens to cause disease. It was shown in earlier studies from this laboratory that the hmuO gene is required for the utilization of iron from heme and hemoglobin in C. diphtheriae. Recent studies have focused on; 1) understanding the molecular mechanisms of hmuO gene regulation, 2) the characterization of factors involved the transport of heme in C. diphtheriae 3) the development of genetic tools in C. diphtheriae to more accurately define the function of various genetic systems and 4)the characterization of a DtxR homolog in C diphtheriae termed MntR. Transcription from the hmuO promoter has been shown to be under dual regulation in which expression is positively regulated by a heme source, such as heme or hemoglobin, and negatively regulated by the diphtheria toxin repressor protein (DtxR) and iron. Studies in this laboratory have shown that heme-dependent activation of the hmuO promoter occurs through a two component signal transduction system that requires the chrA and chrS genes, which encode a response regulator and sensor kinase, respectively. An ABC (ATP-binding cassette) type transport system is involved in the utilization of heme and hemoglobin as iron sources in C. diphtheriae and the HmuT lipoprotein protein is proposed to be the cell surface heme receptor. A system for constructing defined mutation was developed for both C. diphtheriae and C. ulcerans. A homolog of dtxR, mntR, was identified from the recently completed C. diphtheriae genome. The mntR gene was cloned and was shown to be the terminal gene in a five gene operon that also encoded a putative ABC-metal transporter. The product of mntR was shown to regulate expression of this operon by a Mn-dependent mechanism.